Project 4 will study the paucibacillary state that occurs during Tuberculosis (TB) treatment. TB must be treated for a minimum of six months to prevent relapses. Prolonged treatment places a huge burden on TB control programs and creates many opportunities for non-compliance, relapse and the emergence of drug resistance. Efforts to improve the speed and effectiveness of TB treatments are hampered by critical gaps in our understanding of the paucibacillary stage of TB. Achieving improved kill kinetics at this stage would enable greatly shortened and more successful treatment. Foremost among these unknowns are 1) the biology of a probably metabolically diverse population of Mycobacterium tuberculosis (Mtb) that can persist in the presence of drugs without being killed (persisters), yet have normal MICs; 2) the bacterial and host pharmacokinetic/pharmacodynamic determinants of bacterial burden during the paucibacillary stage of TB treatment; and 3) the relationship between these bacterial and host factors and clinical relapse. Our group has identified a novel set of Mtb mutants with altered persistence. We have also identified clinical Mtb strains that are more prone to relapse after TB treatment, and found that they may have a new persister phenotype related to altered gene expression and moderately higher MICs against TB drugs. We have also noted altered drug pharmacokinetics within TB lesions that can decrease drug delivery and interact with bacterial MIC to decrease bacterial killing. To find ways to more rapidly overcome persistence during TB treatment, we must first understand all the bacterial and host elements that contribute to it. In this project, we will 1) identify persistence mechanisms relevant to human TB treatment; 2) determine whether Mtb strains with moderately increased (but drug-susceptible) MICs are more prone to relapse after treatment, and identify the underlying mechanism of these MIC differences along with useful predictive biomarkers; 3) examine In vivo relevance of persistence mutants in rabbit disease model and effect of lesional PK/MIC ratios on lesion-centric efficacy (kill rates). A full understanding of these factors and how they interact to determine the fate of Mtb bacilli remaining in host tissues during treatment is critical to developing shorter therapies